Pulse radiolysis study of a yeast cytochrome c from Hansenula anomala

The reduction of Hansenula anomala yeast cytochrome c by e-aq and CO-.2 was investigated by pulse radiolysis, at a high reductant to protein concentration ratio. The reactivity of the radicals was studied by observing absorbance changes in the cytochrome c spectrum over the wavelength range 280-600 nm. At pH 7, over the time scale of the radical decays (i.e. 0-4 microseconds for e-aq; 0-40 microseconds for CO-.2s) and beyond, the hemoprotein was reduced without any spectrally detected intermediate between ferri-and ferro-forms. This conclusion was reached by simulation studies based on the direct reduction of the yeast cytochrome c from the ferri- to the ferro-form, yielding a correct fit between experimental and calculated absorbance curves. The reduction rate constants were determined to be 1.0 +/- 01 X 10(10) M-1 S-1 for e-aq and 0.7 +/- 0.05 X 10(9) M-1 S-1 for CO-.2 at 0.16 M ionic strength, pH 7.0 and 20 degrees C, thus not significantly different from other values reported for horse heart cytochrome c. However, in the 360-390 nm region the generation of an additional radical species was noticed. The present experimental data were compared with previously published reports.     

Gating current kinetics in Myxicola giant axons. Order of the back transition rate constants.

Gating current, Ig, was recorded in Myxicola axons with series resistance compensation and higher time resolution than in previous studies. Ig at ON decays as two exponentials with time constants, tau ON-F and tau ON-S, very similar to squid values. No indication of an additional very fast relaxation was detected, but could be still unresolved. Ig at OFF also displays two exponentials, neither reflecting recovery from charge immobilization. Deactivation of the two I(ON) components may proceed with well-separated exponentials at -100 mV. INa tail currents at OFF also display two exponentials plus a third very slow relaxation of 5-9% of the total tail current. The very slow component is probably deactivation of a very small subpopulation of TTX sensitive channels. A -100 mV, means for INa tail component time constants (four axons) are 76 microseconds (range: 53-89 microseconds) and 344 microseconds (range: 312-387 microseconds), and for IOFF (six axons) 62 microseconds (range: 34-87 microseconds) and 291 microseconds (range: 204-456 microseconds) in reasonable agreement. INa ON activation time constant, tau A, is clearly slower than tau ON-F at all potentials. Except for the interval -30 to -15 mV, tau A is clearly faster than tau ON-S, and has a different dependency on potential. tau ON-S is several fold smaller than tau h. Computations with a closed2----closed1----open activation model indicated Na tail currents are consistent with a closed1----open rate constant greater than the closed2----closed1.     

Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels

An electrochemical method for the simultaneous detection of two different tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP), in one-pot, using CdS/DNA and PbS/DNA nanochains as labels was developed. Herein, magnetic beads (MBs) as bimolecule immobilizing carriers, were used for co-immobilization of primary anti-CEA and anti-AFP antibodies. The distinguishable signal labels were synthesized by in situ growth of CdS and PbS nanoparticles on DNA chains, respectively, which were further employed to label the corresponding secondary antibodies. A sandwich-type immunoassay format was formed by the biorecognition of the antigens and corresponding antibodies. The assay was based on the peak currents of Cd(2+) and Pb(2+) dissolved from CdS and PbS nanoparticles by HNO(3) using square wave stripping voltammetry. Experimental results show that the multiplexed electrochemical immunoassay has enabled the simultaneous monitoring of CEA and AFP in a single run with wide working ranges of 0.1-100ngmL(-1) for CEA and 0.5-200ngmL(-1) for AFP. The detection limits reach to 3.3pgmL(-1) for CEA and 7.8pgmL(-1) for AFP.     

Limits of phase and amplitude sensitivity in the torus semicircularis ofEigenmannia

Eigenmannia can detect modulations in the time disparity of signals received by different regions of the body surface as small as several hundred nanoseconds. This study presents recordings of single units in the torus semicircularis that are sensitive to time disparities (differential-phase) between a sinusoidal signal received by the head region and a similar signal received by the body surface caudal to the fish's pectoral fins. The sensitivity of units to differential phase, measured by the change in spike rate per unit change in time disparity, was greatest when small phase modulations, rather than stationary phase differences, were presented. Thresholds of differential-phase coders ranged from 6.5 microseconds to several hundred microseconds, with approximately 20% of the units having thresholds in the 5-10 microseconds range. For most cells, sensitivity to small modulations of differential-phase was relatively unaffected by time disparity 'offsets' within a range of several hundred microseconds. A threshold of 5-10 microseconds is still an order of magnitude higher than that measured in the Jamming Avoidance Response (JAR). Neurons that were sensitive to amplitude modulations (AMs) had thresholds as low as 0.05%. This value is comparable to that observed at the behavioral level.     

Generation of fluorescence quenchers from the triplet states of chlorophylls in the major light-harvesting complex II from green plants

Laser flash-induced changes of the fluorescence yield were studied in aggregates of light-harvesting complex II (LHCII) on a time scale ranging from microseconds to seconds. Carotenoid (Car) and chlorophyll (Chl) triplet states, decaying with lifetimes of several microseconds and hundreds of microseconds, respectively, are responsible for initial light-induced fluorescence quenching via singlet-triplet annihilation. In addition, at times ranging from milliseconds to seconds, a slow decay of the light-induced fluorescence quenching can be observed, indicating the presence of additional quenchers generated by the laser. The generation of the quenchers is found to be sensitive to the presence of oxygen. It is proposed that long-lived fluorescence quenchers can be generated from Chl triplets that are not transferred to Car molecules. The quenchers could be Chl cations or other radicals that are produced directly from Chl triplets or via Chl triplet-sensitized singlet oxygen. Decay of the quenchers takes place on a millisecond to second time scale. The decay is slowed by a few orders of magnitude at 77 K indicating that structural changes or migration-limited processes are involved in the recovery. Fluorescence quenching is not observed for trimers, which is explained by a reduction of the quenching domain size compared to that of aggregates. This type of fluorescence quenching can operate under very high light intensities when Chl triplets start to accumulate in the light-harvesting antenna.     

Synthesis of a small library of bivalent α-D-mannopyranosides for lectin cross-linking

A small library of bivalent α-D-mannopyranosides having rigid linkers was constructed in order to evaluate the effects of inter-saccharide distances upon multivalent binding interactions with plant and bacterial lectins. To this end, iodoaryl and propargyl α-D-mannopyranosides were synthesized and the former treated with TMS-acetylene under palladium chemistry to provide their corresponding ethynylaryl derivatives. A library of 15 dimeric members was then obtained using Lewis acid catalyzed glycosidation, aryl-aryl homocoupling, transition metal catalyzed Sonogashira cross-coupling reactions, and oxidative Glaser homocoupling.     

Synthesis of a small library of bivalent α-d-mannopyranosides for lectin cross-linking

A small library of bivalent α-d-mannopyranosides having rigid linkers was constructed in order to evaluate the effects of inter-saccharide distances upon multivalent binding interactions with plant and bacterial lectins. To this end, iodoaryl and propargyl α-d-mannopyranosides were synthesized and the former treated with TMS-acetylene under palladium chemistry to provide their corresponding ethynylaryl derivatives. A library of 15 dimeric members was then obtained using Lewis acid catalyzed glycosidation, aryl–aryl homocoupling, transition metal catalyzed Sonogashira cross-coupling reactions, and oxidative Glaser homocoupling.     

Fast repair of hydroxy radical purine deoxynucleotide adducts by phenylpropanoid glycosides and their derivatives from Chinese herbs

DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very significant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells' inadequate repair capacity. The repair activities and reaction mechanism of phenylpropanoid glycosides (PPGs) and their derivatives, isolated from Chinese folk medicinal herbs, towards both dGMP-OH* adducts and dAMP-OH* adducts were studied with the pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mM dGMP or dAMP aqueous solution containing one of the PPGs or their derivatives, the transient absorption spectra of the hydroxyl adduct of dGMP or dAMP decayed with the formation of that of phenoxyl radicals of PPGs or their derivatives within several decades of microseconds after electron pulse irradiation. The result indicated that dGMP or dAMP hydroxyl adducts can be repaired by PPGs or their derivatives. The rate constants of the repair reactions were deduced to be 0.641-1.28 x 10(9) M(-1) s(-1) for dGMP-OH* and 0.2-0.491 x 10(9) M(-1) s(-1) for dAMP-OH*, which positively correlated to the number of phenolic hydroxyl groups in the glycoside structure. A deeper understanding of this new repair mechanism may help researchers to design strategies to prevent and/or intervene more effectively in free radical related diseases.     

Vibrational spectroscopy to study the properties of redox-active tyrosines in photosystem II and other proteins

Tyrosine radicals play catalytic roles in essential metalloenzymes. Their properties--midpoint potential, stability...--or environment varies considerably from one enzyme to the other. To understand the origin of these properties, the redox tyrosines are studied by a number of spectroscopic techniques, including Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopy. An increasing number of vibrational data are reported for the (modified-) redox active tyrosines in ribonucleotide reductases, photosystem II, heme catalase and peroxidases, galactose and glyoxal oxidases, and cytochrome oxidase. The spectral markers for the tyrosinyl radicals have been recorded on models of (substituted) phenoxyl radicals, free or coordinated to metals. We review these vibrational data and present the correlations existing between the vibrational modes of the radicals and their properties and interactions formed with their environment: we present that the nu7a(C-O) mode of the radical, observed both by RR and FTIR spectroscopy at 1480-1515 cm(-1), is a sensitive marker of the hydrogen bonding status of (substituted)-phenoxyl and Tyr*, while the nu8a(C-C) mode may probe coordination of the Tyr* to a metal. For photosystem II, the information obtained by light-induced FTIR difference spectroscopy for the two redox tyrosines TyrD and TyrZ and their hydrogen bonding partners is discussed in comparison with those obtained by other spectroscopic methods.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

The rise times of miniature endplate currents suggest that acetylcholine may be released over a period of time.

Models of miniature endplate currents predict 20-80% rise times of 100 microseconds or less. These predictions are substantially less than most of the rise times recorded in the literature. New measurements were made of rise times at the frog neuromuscular junction using extracellular recording. The mean 20-80% rise time was 250 microseconds. Rise times were variable; at 20 degrees C, 95% of them fell in a range from 140 to 460 microseconds. The most questionable assumption in the models is that the acetylcholine (ACh) is released instantaneously. Modifying the model, so that ACh diffuses from the vesicle through a pore, lengthens the rise time to observed levels. It has been proposed that ACh is released from the vesicle in exchange for Na+. However, the rise times of miniature endplate currents recorded in solutions in which the Na+ is replaced by sucrose are in the normal range. The Q10 for the rise of miniature endplate currents is approximately 2, which is consistent with the models and with temperature effects on pore formation in mast cells.     

Support-controlled chemoselective olefin-imine addition photocatalyzed by cadmium sulfide on a zinc sulfide carrier.

The semiconductor catalyzed photoaddition of cyclopentene or cyclohexene to various novel electron-poor imines of type p-XC(6)H(4)(CN)C[double bond, length as m-dash]N(COPh) (X = H, F, Cl, Br, Me, MeO) was investigated as a function of the nature of the cadmium sulfide photocatalyst. Irradiation (lambda>/= 350 nm) of silica supported cadmium sulfide surprisingly did not afford the expected olefin-imine adducts but an imine hydrocyanation product via an unprecedented dark reaction. However, when silica was replaced by zinc sulfide as the support for cadmium sulfide, the expected homoallylic N-benzoyl-alpha-amino cyanides were isolated in yields of 65-84%. Thus, chemoselectivity is introduced through replacing an insulating by a semiconducting support, a hitherto unknown effect in semiconductor photocatalysis. From the sign of the time resolved photovoltage it is found that the mixed metal sulfide interface CdS/ZnS increases the lifetime of photogenerated electron-hole pairs by about one order of magnitude as compared to the SiO(2)/CdS system. The reaction rate increases with increasing imine sigma-Hammett constants and decreasing stability of intermediate benzyl radicals.     

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded oligonucleotides

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.     

Opening and closing transitions for BK channels often occur in two steps via sojourns through a brief lifetime subconductance state.

Single channel currents were recorded with microsecond time resolution from large-conductance calcium-activated K+ channels to examine the details of the opening and closings transitions. Analysis of averaged closing transitions indicated that the initial average conductance step for closing was to the 90-95% closed channel current level. Averaged brief closings (approximately 50 microseconds) reopened from the initial 90-95% level, whereas averaged longer closings (> 300 microseconds) closed completely from this level over the next 50-100 microseconds. The 90-95% initial closed level in the averaged current records resulted typically from the average of both complete and partial closings. From 45-80% of the initial closings were complete and 20-55% were to brief lifetime (approximately 50 microseconds) subconductance levels at 65-90% of the completely closed level. Averaged opening transitions were typically mirror images of averaged closing transitions. To extend the analysis to the very brief conductance changes that underlie the flickers of the single channel current toward the closed current level, flickers, brief closings, and longer closings were averaged separately and their slopes compared. The slopes were similar (within the 3% resolution of the method), suggesting similar initial conductance steps. Similar initial closing properties for both the briefer and longer closings would be expected if the channel first passed through the kinetic and subconductance states associated with the briefer closings (including flickers) before entering the longer closed states. Such transitions would provide an explanation for the observation that openings and closings often occur in two steps.     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediatedperoxidation of phosphatidyl choline liposomes

Abstract

The antioxidant efficacy of α-carotene and comparison with β-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2′-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS)at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of α- and β-carotenes, and the chain breaking antioxidant α-tocopherol were also included in the study.AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples.In this system, α-carotene retarded PCOOH formation better than β-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than α- and β-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, α-, β-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85%and 82%, respectively, while α-tocopherol elicited 90%reduction.AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P <0.001). α-Carotene significantly suppressed the TBARS formation by 78% whilst β-carotene, lutein, zeaxanthin and α-tocopherol elicited 60%, 91%and 80% reductions, respectively. Increasing the concentration of the carotenoid >1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack.Our findings suggest that α-carotene is a better antioxidant than is β-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids _{in vivo}.     

A mixed-ligand iron-sulfur cluster (C556SPaB or C565SPsaB) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I.

The proposed structure of Photosystem I depicts two cysteines on the PsaA polypeptide and two cysteines on the PsaB polypeptide in a symmetrical environment, each providing ligands for the interpolypeptide Fx cluster. We studied the role of Fx in electron transfer by substituting serine for cysteine (C565SPsaB and C556SPsaB), thereby introducing the first example of a genetically engineered, mixed-ligand [4Fe-4S] cluster into a protein. Optical kinetic spectroscopy shows that after a single-turnover flash at 298 K, the contribution of A1- (lifetime of 10 microseconds, 40% of total and lifetime of 100 microseconds, 20% of total) and Fx- (lifetime of 500-800 microseconds, 10-15% of total) to the overall P700+ back reaction have increased in C565SPsaB and C556SPsaB at the expense of the back reaction from [FA/FB]-. The electron paramagnetic resonance spectrum of Fx shows g-values of 2.04, 1.94, and 1.81 in both mutants and a similarly decreased amount of FA and FB reduced at 15 K after a single-turnover flash. These results indicate that the mixed-ligand (3 cysteines, 1 serine) Fx cluster is an inefficient electron carrier, but that a small leak through Fx still permits FA and FB to be reduced quantitatively when the samples are frozen during continuous illumination. The data confirm that Fx is a necessary intermediate in the electron transfer pathway from A1 to FA and FB in Photosystem I.     

Effective discrimination of antimalarial potency of artemisinin compounds based on quantum chemical calculations of their reaction mechanism

The reaction mechanism of 12 antimalarial artemisinin compounds with two competitive pathways was studied by means of quantum chemical calculations using the IMOMO(B3LYP/6-31(d,p):HF/3-21G) method. The oxygen-centered radicals, carbon-centered radicals, and transition states (TS) in both pathways were geometrically optimized. The obtained kinetic and thermodynamic energy profiles show that homolytic C-C cleavage reaction (pathway 2) is energetically more preferable than an intramolecular 1,5-hydrogen shift process (pathway 1), which is consistent with the docking calculations. However, compounds that can easily proceed along the pathway 1 have high activity. Therefore, both pathways are important for antimalarial activity. Moreover, effective discrimination between high and low activity compounds using EA1, deltaE1, and deltaE(1A-2A) was accomplished.     

Further Investigation on Naturally Occurring Tocopherols and Tocotrienols in Bovine Milk Fat

The polar lipid material which contains most of unsaponiriable matter of milk fat was collected by means of neutral alumina column chromatography. After saponification of the polar lipid material, the unsaponiriable matter was purified by repeated Florisil and neutral alumina column chromatography and the total tocopherol fraction was obtained. It was found that the total tocopherol fraction isolated from milk fat contained 6 of the known naturally occurring tocopherols, that is, α-, β-, γ-, and δ-tocopherols and α- and γ-tocotrienols. These were identified by two-dimensional thin-layer and gas-liquid chromatography before and after hydrogenation.     

Bystander cell death and stress response is inhibited by the radical scavenger α(1)-microglobulin in irradiated cell cultures

Alpha-particle irradiation of cells damages not only the irradiated cells but also nontargeted bystander cells. It has been proposed that the bystander effect is caused by oxidants and free radicals generated by the radiation. Recent studies have shown that α(1)-microglobulin protects against cell damage caused by oxidants and free radicals. Using a novel experimental system that allows irradiation of 0.02% of a human hepatoma monolayer, leaving 99.98% as bystander cells, we investigated the influence of oxidative stress and the cell-protective effects of α(1)-microglobulin during α-particle irradiation. The results showed an increase in cell death in both irradiated cells and bystander cells. A significant increase in apoptosis, oxidation markers and expression of the stress response genes heme oxygenase 1, superoxide dismutase, catalase, glutathione peroxidase 1, p21 and p53 were observed. Addition of α(1)-microglobulin reduced the amount of dead cells and inhibited apoptosis, formation of oxidation markers, and up-regulation of stress response genes. The results emphasize the role of oxidative stress in promoting bystander effects. Furthermore, the results suggest that α(1)-microglobulin protects nonirradiated cells by eliminating oxidants and free radicals generated by radiation and imply that α(1)-microglobulin can be used in radiation therapy of tumors to minimize damage to surrounding tissues.     

Formation of cyclodextrin inclusion compounds of stable nitroxide radicals as monitored by electron-spin resonance spectra

Hyperfine coupling constants and rotational correlation times, calculated from electron-spin resonance spectra of cyclodextrins incubated with stable nitroxide radicals, indicate inclusion compound formation of β- and γ-cyclodextrin with certain nitroxide radicals. In contrast, α-cycledextrin exhibits no effect on the spectra of the radicals, probably because its central cavity is too small to form such inclusion compounds. Furthermore, one 1:1 molar ratio complex of β-cyclodextrin and a nitroxide radical (isolated as crystalline precipitate and identified both by combustion analysis and ir measurements) is shown by electron-spin resonance data to be an inclusion compound.